Clostridial toxins are responsible for
severe diseases in man and animals such as botulism,
gangrenes and necrotic enteritis. Our laboratory is involved
in the study of the regulation of the toxin synthesis
in Clostridium botulinum and Clostridium tetanu, and of the mode of action of clostridial toxins
such as lethal toxin from Clostridium sordellii.

The passage of BoNT/A
trough the intestinal barrierwas
investigated in polarized intestinal cell monolayers grown on
filters. BoNT/A crosses intestinal cell monolayers via
a receptor-mediated transcytosis, including a transport
inhibition at 4°C and a passage at 37°C in a
saturable manner within 30-60 min. BoNT/A passage rate was
about 10-fold more efficient through the intestinal crypt
cell line m-ICcl2, than through the carcinoma
Caco-2 or T-84 cells, and was not increased when BoNT/A was
associated with the non-toxic proteins (botulinum complex).
Like for neuronal cells, BoNT/A binding to intestinal cells
was mediated by the half C-terminal domain as tested by
immunofluorescence (Fig. 1), fluorescence activated cytometry
and by transcytosis competition assay. Gangliosides of
GD1band GT1bseries and recombinant
intravesicular SV2-C domain partially impaired BoNT/A
transcytosis, suggesting a putative role of gangliosides and
SV2 or a related protein in BoNT/A transcytosis through
Caco-2 and m-ICcl2cells.

Large clostridial
toxinssuch as LT from
C.
sordellii glucosylates various Rho
and Ras GTPases. We have found that LT binds specifically to
phosphatidylserine (PS) and that LT glucosylation activity is
higher when the substrate Rac is linked to lipid membrane,
preferentially enriched in PS. LT preferentially alters
basolateral actin and E-cadherin intercellular junctions of
polarized epithelial cells, induces apoptosis in myeloid cell
line, as well as degeneration and regeneration of skeletal
neuromuscular tissue. Lethal activity of LT was investigated
in mouse. LT induces a massive extravasation of blood
fluid in the thoracic cage, resulting from an increase in
lung vascular permeability, which generates profound
modifications such as animal dehydration, increase in
hematocrit, hypoxia (as assessed by the increase in serum
erythropoietin), and, finally, cardio-respiratory distress.
Immunohistochemical analysis demonstrates that in lung
endothelial cells, VE-cadherin, a protein participating in
intercellular adherens junctions, is redistributed from
membrane to cytosol. No major sign of inflammation was
induced by LT. Currently, the LT-dependent
cellular pathways between inactivation of Rho/RasGTPases and
actin depolymerization are investigated.

Characterization of new
toxins and Clostridiumdiffiicile variants.We have
characterized a type III secreted toxin by
Aeromonas
salmonicida called AexT which alters
the actin cytoskeleton. AexT catalyzes an ADP-ribosylation
reaction with monomeric actin, as clostridial binary toxins
do, and possesses a GTPase activity (GAP) towards Rho, Rac,
and Cdc42, which are involved in the control of the assembly
of actin filaments. AexT is the first toxin to be described
that has a specific actin ADP-ribosylation activity and GAP
activity towards Rho, Rac and Cdc42, both activities
contributing to actin filament depolymerization.

Some C.perfringenstype B and C strains produce
an additional toxin called Delta toxin, which has been found
to be hemolytic and cytotoxic for cells expressing the
ganglioside GM2in their membrane. We have reported
the genetic characterization of Delta toxin and the
production of active recombinant protein, and shows that its
mode of action consists in pore formation in lipid bilayer.
Although Delta toxin shows a significant homology with Beta
toxin, both toxins recognize distinct cell surface receptors
and differ by the properties of channels formed in lipid
bilayers.

A triple-locus nucleotide sequence analysis based on toxin
regulatory genes tcdC, tcdR,and cdtRwas
investigated to characterize the genetic variability of C.
difficileisolates. Five lineages have been distinguished,
which correlate with the different types of deletion in
tcdC. This study evidences the clonality of isolates
(PCR ribotype 027, PCR ribotype 078, and toxin A-negative
toxin B-positive isolates, that are frequently associated
with human infections and food animals.